In the 1920s Otto Warburg first proposed non-oxidative metabolism of glucose as a unique feature of tumors (Warburg, (1930) Ueber den stoffwechsel der tumoren (Lon-don: Constable); Warburg, (1956) Science 123, 309-314). This hypothesis has since caused significant interest and although mechanistic links are still, almost 100 years later, under investigation. A high glucose flux of tumor is today exploited clinically, using PET imaging of 18F-2-deoxyglucose uptake as a diagnostic tool for solid tumors.
Lately, energy processing of cancer cells has been given new attention (e.g. Vander Heiden, et al., 2009, Science 324, 1029). The hypoxic microenvironment and conse-quential lactate accumulation resulting from altered tumor metabolism are reported predictive for both metastatic potential and therapy resistance, and thus survival of cancer patients (Brown, (1999) Cancer Res. 59, 5863-5870; Walenta & Mueller-Klie-ser, (2004) Semin. Radiat. Oncol. 14, 267-274; Walenta et al., (2004) Curr. Med. Chem. 11, 2195-2204). Targeting of hypoxic and/or acidotic tumor areas has therefore drawn attention as a complement to anti-proliferative treatments (see e.g. Pan &Mak, (2007) Sci. STKE 381, pe14; Bache et al., (2008) Curr. Med. Chem. 15, 322-338 for reviews).
Known inhibitors of glycolysis include among others 2-deoxyglucose and 2-bromo-puruvate targeting hexokinase (Liu et al., (2001) Biochemistry 40, 5542-5547; Liu et al. (2002) Biochem. Pharmacol. 64, 1745-1751; Xu et al., (2005) Cancer Res. 65, 613-621; Ramanathan et al., (2005) Proc. Natl. Acad. Sci. USA 102, 5992-5997). Fructose-2,6-bisphosphate (F-2,6-P2) plays a regulatory role in glucose metabolism by relieving ATP inhibition of phosphofructokinase-1. The levels of F-2,6-P2 are regulated by the bifunctional enzyme family 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1-4).
Out of these four isozymes, mainly PFKFB3 and PFKFB4 are of particular interest for playing a role in cancer. Anti-sense treatment against PFKFB3 was shown to reduce tumor growth rate in vivo (Chesney et al., (1999) Proc. Natl. Acad. Sci. USA 96, 3047-3052). Similarly, a decreased anchorage independent growth was shown for siRNA treated fibroblasts (Telang et al., (2006) Oncogene 25, 7225-7234). A link between inflammation and enhanced glycolysis and a possible potential for PFKFB3 inhibitors to act as a anti-inflammatory agents was indicated by a report that the IL-6-STAT3 pathway may enhance glycolysis through the induction of PFKFB3 (Ando et al. J Nippon Med Sch (2010), 77, (2), 97-105). This possibility was further supported by a re-cent study using a small molecule; 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), previously shown to reduce F-2,6-P2 synthesis, glucose uptake and proliferation in transformed cells (see below). Telang et al. demonstrated that 3PO attenuates the activation of T cells in vitro and suppresses T cell dependent immunity in vivo, indicating that small molecule inhibitors of PFKFB3 may prove effective as T cell immunosuppressive agents (Telang et al., (2012) Journal of Translational Medicine 2012, 10:95). Moreover, hypoxia is a prominent feature in rheumatoid arthritis (RA) synovium and induces significant changes in the expression of PFKFB3 and PFKFB4 (Del Rey et al., (2010) Arthritis & Rheumatism 62, 3584-3594).
The PFKFB4 protein was reported to be strongly responsive to hypoxia (Minchenko et al., (2004) FEBS Lett. 576, 14-20; Minchenko et al., (2005), Biochemie 87, 1005-1010; Bobarykina et al., (2006), Acta Biochemica Polonica 3, 789-799). US2010/0267815 A1). Minchenko et al. demonstrated an increased expression of PFKFB4 mRNA in malignant breast and colon cancers, as compared to corresponding non-malignant tissue counterparts. Recently, Telang et al. showed decreased levels of F-2,6-P2 and lactate as well as decreased tumor growth following siRNA silencing of PFKFB4 (Telang, S. et al, (2010). Further support for PFKFB4 as a potential target for the development of antineoplastic agents came from a functional metabolic screen that identified PFKFB4 as an important regulator in prostate cancer (Ros et al. (2012) Cancer Discov. 2(4):328-43).
Only a small number of specific inhibitors of the kinase activities of PFKFB3 and PFKFB4 have been identified. In one study, an alkylating inhibitor, N-bromoacetylethanolamine phosphate, was used as a tool to investigate the binding sites of the kinase and phosphatase domains of PFKFB3 and demonstrated to irreversibly inactivate PFK-2 (Sakakibara et al. (1984), J. Bio Chem 259, 14023-14028). The compound is a competitive inhibitor of PFK-2 with respect to F6P but a non-competitive inhibitor with respect to ATP. Analogues of this compound, N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide and N-(3-methoxypropyl)-bromoacetamide, have demonstrated in vivo activity with increased survival rate of P388 transplant BDF1 mice (Hirata et al. (2000) Biosci. Biotechnol. Biochem. 64, 2047-2052).
A crystal structure of the PFKFB3*ADP*phosphoenolpyruvate complex was described by Kim et al. (Kim et al. (2007), J. Mol. Biol. 370, 14-26). This paper also described the crystal structures of PFKFB3*AMPPCP*fructose-6 phosphate complex in which β,γ-methylene-adenosine 5′-triphosphate (AMPPCP) constituted a non-hydrolysable ATP-analogue. Recently, small molecule PFKFB3 inhibitors identified by virtual screening were described (Chrochet et al. (2011), Anal. Biochem. 418, 143-148; Seo et al., (2011), Plosone, 9, e24179 & Lee et al. (2012) US 2012/0302631). The identified PFKFB3 inhibitors were shown to reduce the levels of F-2,6-P2, resulting in decreased tumor growth and increased cell death.
A drug-like compound was described (Clem et al. (2008) Mol. Cancer Ther. 7, 110-120; Chesney et al. (2008) WO 2008/156783) where 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), by computational methods, was identified as a PFKFB3 inhibitor. Administration of 3PO reduced the intracellular concentration of F-2,6-P2, glucose uptake, and growth of established tumors in vivo. Recently, substituted benzindoles were described as inhibitors of PFKFB3. The benzindoles were shown to inhibit proliferation in several cancer cell lines, inhibit glucose uptake as well as to reduce tumor growth in vivo in tumor models (Chand et al. (2011) WO2011/103557A1).
Radiotherapy is one of the most efficient tools in treatment of cancer: about 50% of all patients receive γ-irradiation. However, radiotherapy struggles with problems of resistance and negative side effects. To improve prognosis and minimize exposure time there is a need to specifically radiosensitize cancer cells. Attractive targets for radiosensitization are factors involved in the DNA damage response since γ-irradiation induces a broad range of DNA damage including modifications of bases and sugars, single-strand breaks, clustered damage and DNA double-strand breaks (DSBs) (Han, W. & Yu, K. N. F. Ionizing radiation, DNA double strand break and mutation. Adv Gen Res 4, 1-13 (2010)).
The DNA DSB is considered as one of the most detrimental DNA lesions and can if repaired incorrectly lead to gross chromosomal rearrangements, genomic instability or cell death (oeijmakers, J. H. Genome maintenance mechanisms for preventing cancer. Nature 411, 366-374, doi:10.1038/35077232 (2001)). The cellular response to DSB induction involves the activation of DNA damage sensing pathways which ensure recruitment of the correct DNA repair factors and triggering of DNA damage sensing checkpoints. Formation of a DSB will result in the immediate binding of the free DNA ends by the MRN complex acting upstream of ATM, a major DNA damage sensing kinase (Rupnik, A., Lowndes, N. F. & Grenon, M. MRN and the race to the break. Chromosoma 119, 115-135, doi:10.1007/s00412-009-0242-4 (2010)). Both MRN and ATM play a central role in phosphorylation of histone variant H2AX that will spread rapidly creating stretches of γH2AX, the phosphorylated form of H2AX, on both sides of the break (Bernstein, K. A. & Rothstein, R. At loose ends: resecting a double-strand break. Cell 137, 807-810, doi:10.1016/j.cell.2009.05.007 (2009)). This will result in recruitment and accumulation of DSB response factors where 53BP1 is one of the key scaffold proteins required for a proficient DSB repair response (Schultz, L. B., Chehab, N. H., Malikzay, A. & Halazonetis, T. D. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol 151, 1381-1390 (2000); Panier, S. & Boulton, S. J. Double-strand break repair: 53BP1 comes into focus. Nat Rev Mol Cell Biol 15, 7-18, doi:10.1038/nrm3719 (2014)).
Two distinct repair pathways are involved in DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the major repair pathway for DSBs, a fast and efficient repair process that is active throughout the cell cycle. Ligation of free DNA ends are promoted by 53BP1 and dependent on repair factors KU70/80, DNAPKcs and LigaseIV (Panier, S. & Boulton, S. J, vide supra; Weterings, E. & Chen, D. J. The endless tale of non-homologous end-joining. Cell Res 18, 114-124, doi:10.1038/cr.2008.3 (2008)). The NHEJ ligation requires blunting of DNA ends and in cases of extensive end-processing the repair pathway is regarded as error prone. HR takes place in late S and G2 phase where the sister-chromatid can be used as template for an error-free repair. The process is dependent on the resection of DNA ends, promoted by BRCA1, and the loading of RPA on resected over-hangs (Sleeth, K. M. et al. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells. J Mol Biol 373, 38-47, doi:10.1016/j.jmb.2007.07.068 (2007)). This initial step is required for RAD51 loading and subsequent strand invasion of the intact homologous sequence (Baumann, P., Benson, F. E. & West, S. C. Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757-766 (1996)).
The DNA repair pathway choice is dependent on cell cycle phase and the cell cycle progression is also tightly regulated in response to DNA damage. The cell division cycle is controlled through phase specific checkpoints that have the ability to arrest cell cycle progression and if necessary, direct cells to undergo apoptosis (Bartek, J. & Lukas, J. DNA damage checkpoints: from initiation to recovery or adaptation. Curr Opin Cell Biol 19, 238-245, doi:10.1016/j.ceb.2007.02.009 (2007)). S-phase cells exhibit enhanced sensitivity to DNA damage since they are in the process of replicating the entire cell genome. Disturbance of the replicative process is collectively known as replication stress and is a major source of genomic instability. Collision of progressing replication forks with DNA damage or repair intermediates can cause severe damage since stalled replication forks are prone to collapse generating DSBs (Groth, P. et al. Methylated DNA causes a physical block to replication forks independently of damage signalling, O(6)-methylguanine or DNA single-strand breaks and results in DNA damage. J Mol Biol 402, 70-82, doi:10.1016/j.jmb.2010.07.010 (2010); Arnaudeau, C., Lundin, C. & Helleday, T. DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 307, 1235-1245, doi:10.1006/jmbi.2001.4564 (2001)). Similarly, exhaustion of replication factors or checkpoint defects can cause de-regulation of replication, stalled replication forks and formation of DSBs (Saintigny, Y. et al. Characterization of homologous recombination induced by replication inhibition in mammalian cells. EMBO J 20, 3861-3870, doi:10.1093/emboj/20.14.3861 (2001); Syljuasen, R. G. et al. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25, 3553-3562, doi:10.1128/MCB.25.9.3553-3562.2005 (2005)). Collapsed replication forks are mainly a substrate for HR repair where deficiency in this repair pathway results in increased sensitivity to replication stress and accumulation of DSBs (Arnaudeau, C., Lundin, C. & Helleday, T., vide supra; Saintigny, Y. et al. vide supra). Factors involved in the DNA repair and replication regulation is of the highest interests as targets in cancer therapy (Helleday, T. DNA repair as treatment target. Eur J Cancer 47 Suppl 3, S333-335, doi: 10.1016/S0959-8049(11)70192-7 (2011)).